VL4: Some molecular methods in infection biology Flashcards
What are some typical questions in infection biology?
-Whar organisms is causing the infection?
-How does it cause the infection?
What are the relevent virulence factors?
Define pathogenicity, virulence and virulence factor
Pathogenicity: ability to cause disease
Virulence: degree of pathogenicity
Virulence factor: cellular factors contributing to virulence
WHich factors are important in vitro, in vivo, virulence?
Essential factors are not always the same in vivo and in vitro, in vivo. virulence factors are not important in vitro, so it is hard to study them in vitro, not all factors that are essential in vivo are essential in vitro (some metabolic pathways enzymes and virulence factors ect…) not all virulence factors are essential in vivo ( QS, secretion systems …
Name methods to study cell biology, DNA, RNA, prot
Cell biology:
- Cell growth
- Microscopy
DNA:
- PCR
- Genome sequencing
- Bioinformatics
RNA:
RT-PCR
cDNA microarrays
Protein -WEster blots Proteomics -Transfections -Chemical genetics
What is the advantage of histology?
You can see changes in healthy/unhealthy tissues
bacteria, fibrin deposition, inflammatory cell infiltrate
Explain the prinziple of blotting
DNA/RNA/Protein sample separated on gel –> transfer to paper, visualize specific bands of interest with probes
Type of blots for which molecule?
Southern blot –> DNA
Northern blot: RNA
Western blot Protein
Explain the principle of PCR?
Denaturation (95°C), Annealing (50-60°C), Extension (68-72°C)
Which ingredients are neede for PCR?
DNA (template), Primers (forward, reverse), DNA polymerase, Salt (MgCl2), dNTPs (ATGC)
What is qPCR used for?
quantify DNA or RNA
- how many copies of a DNA sequence in genome?
- how many RNA transcripts of a gene?
- How many copies of a dna or rna in a sample?
quantification usually relative to control standard, but also absolute quantification
What are Ct value and threshold?
Ct= number of cycles required to reach threshold
threshold is amount of fluorescence required to detect sample/fluorescence above background
the lower the ct the more abundant the inital target
What type of primers can be used for qRT-PCR
specific
random hexamer
polyT
cDNA used a template for PCR
How does the TaqMan method foor qRT-PCR work?
tagman probe bind to sequence, gets altered when polymerization reaches it –fluorescene changes
How does CYBR-Green qRT-PCR work?
sybr green is a dye than caan intercalate into dann and fluoresces upon light excitation when bound to double stranded dann
sample becomes more fluorescent during extension
How can qrtpcr be quantified (absolute)?
use of a Dilution series with known number of copiesto create a Standard curve on which the dna Content can be read with the ct value
Which 2 qualifiers are important when chocing which sequencing technique to be used?
read length
reads/run
What is deep sequencing? (NGS)
sequencing of genomes of a Population of cells
see how populations evolve, see variants arise and compete, to study metagenomes (gut soil ect)
How do cDNA microarrays work and what are they used for?
Arrangement of cDNA on microscopy slide,
let cells grow on Glucose or Ethanol medium, Isolate total mrna, reverse transcribe to cdna labeld with a fluorescent dye, mix, hybridize to dann microarray, wash, measure green and red fluorescence over each spot
quantify rRNA Levels
How can RNA be sequenced and quantified?
mrna Isolation sequencing align sequences against Genome generate sequence Counts for all genes in Genome gene A 30/10 = 3 fold Change gene B 10/5 = 2 fold change
4 immuno basedmethods for alaysis
immunofluorescence (prot and cells) (ab recognition sex ab with fluorescent probe
FACS fluorescence assosiated cell sorting
ELISA (prot)
Western blot, recognition of prot on Membrane with prim, sec ab
How does 2D Gel electrophoresis work?
- isoelectric focusing gel -> on sds plyacrylamide gel (sep by mass)
after Separation identification with diff methods, prot sequencing
What is mass spectrometry and what is it good for?
measuring the mass of Atoms in a molecule
identification of prot
can be done with diff Labels and tags
How can B be identified? (MALDI-TOF MS, pyrosequencing)
malditof uses mass spectromety on bac samples to identify species
compares unknown sample to data of known samples
or Pyrosequencing: Cell culture, Isolate dnaa pcr pyrosequencing with dTTP, programs gets fluorescent Signals for diff nucleotides
identification of species sequence genome
